Fluorescence imaging can map the electrical activity and communication of multiple spatially resolved neurons and thus complement traditional electrophysiological measurements (1, 2). Ca2+ imaging is the most popular of such techniques, because the indicators are well-developed (3-6), highly sensitive (5, 6), and genetically encodable (7-13), enabling investigation of the spatial distribution of Ca2+ dynamics in structures as small as dendritic spines and as large as functional circuits. However, because neurons translate depolarizations into Ca2+ signals via a complex series of pumps, channels, and buffers, fluorescence imaging of Ca2+ transients cannot provide a complete picture of electrical activity in neurons. Observed Ca2+ spikes are temporally low-pass filtered from the initial depolarization and provide limited information regarding hyperpolarizations and subthreshold events. Therefore, while fluorescence imaging of Ca2+ can provide spatial resolution at the sub- and supra-cellular level, the signals are downstream of the action potential, difficult to resolve in fast spiking neurons, buffered by the indicators themselves, and biased toward post-threshold events.
Direct measurement of transmembrane potential with fluorescent indicators would provide a more accurate account of the timing and location of neuronal activity. Despite the promise of fluorescent voltage-sensitive dyes (VSDs), previous classes of VSDs have been hampered by some combination of insensitivity, slow kinetics (14-16), heavy capacitative loading (17-21), lack of genetic targetability, or phototoxicity. Two of the more widely used classes of VSDs, electrochromic and FRET dyes, illustrate the problems associated with developing fast and sensitive fluorescent VSDs.
Electrochromic dyes respond to voltage through a direct interaction between the chromophore and the electric field (FIG. 1A). This Stark effect leads to small wavelength shifts in the absorption and emission spectrum. Because the electric field directly modulates the energy levels of the chromophore, the kinetics of voltage sensing occur on a timescale commensurate with absorption and emission, resulting in ultrafast (fs to ps) hypso- or bathochromic shifts many orders-of-magnitude faster than needed to resolve fast spiking events and action potentials in neurons. The small wavelength shift dictates that the fluorescence signal can be best recorded at the edges of the spectrum, where intensity varies most steeply as a function of wavelength. The largest linear responses are −28% ΔF/F per 100 mV (22), although more typical values are ˜10% per 100 mV (23, 24). Photo-induced electron transfer (PeT)-based Ca2+ probes, such as fluo-3, give ΔF/F values of up to 150% for action potentials in cultured hippocampal neurons (25). Therefore, although electrochromic dyes can keep pace with fast voltage oscillations in neurons, their insensitivity limits the systems in which these dyes can successfully report on voltage changes.
FRET-based voltage sensors use lipophilic anions that intercalate into the cellular membrane and redistribute between the inner and outer leaflets depending upon the transmembrane potential (FIG. 1B). The Nernstian distribution is monitored by a second fluorophore immobilized on one side of the membrane, which undergoes FRET preferentially with the mobile anions on the same side of the membrane. Translocation of the lipophilic anion through the lipid bilayer governs the kinetics of voltage sensing, which can be in the millisecond range. Although these two-component systems can give large changes in intensity (5-34%) (21) or ratio (80% per 100 mV) (15), the operative mechanism relies on translocation of mobile charges in the plasma membrane, thus introducing a capacitative loading problem and resulting in slow response times.
In view of the above drawbacks, methods and compositions are needed which are sensitive to small variations in transmembrane potentials and can respond both to rapid, preferably on a millisecond timescale, and sustained membrane potential changes. Also needed are methods and compositions less susceptible to capacitative loading issues and capable of providing a ratiometric fluorescence signal. The present invention fulfills these and other needs.